A major issue in developmental biology is to determine how time and position-restricted instructions are signaled and received during morphogenesis of different phenotypes, of which tooth, Meckel's cartilage and tongue formation are classical examples. It is now evident that a hierarchy of growth factors and their downstream transcription factors regulate the timing, sequence and position of cells and tissues in forming different phenotypes during embryogenesis (Edelman, 1985; Lumsden and Krumlauf, 1996). We have developed an organ culture model in which mandibular explants formed tooth bud, Meckel's cartilage, tongue and osteoid-like tissue (Slavkin et al., 1989; Chai et al., 1994, 1997). Using this model, our studies have shown that transforming growth factor-beta (TGF-beta) ligands are expressed in a time and tissue specific manner and play important regulatory roles during first branchial arch morphogenesis (Chai et al., 1994). Recently, abundant molecular evidence has demonstrated that both TGF-beta type I and type II (IR and IIR) receptors as well as their downstream mediators (Smad2, Smad3 and Smad4) are required components of the TGF-beta signaling pathway (Massague, 1996). This research proposal is based on the hypotheses that (1) TGF-beta IR, IIR, Smad2, Smad3 and Smad4 are required for the signaling of TGF-beta ligands which regulate morphogenesis of the first branchial arch during craniofacial development. (2) the regulation of morphogenesis by TGF-beta is mediated by an intracellular signaling pathway that down regulates transcription factor Lef1. Three Specific Aims are designed to test the hypotheses: (1) to determine and compare the temporal and spatial expression of TGF- beta IR, IIR, Smad2, Smad3 and Smad4 during the formation of tooth, Meckel's cartilage and tongue both in vivo and in vitro using in-situ hybridization, RT-PCR, and immunohistochemistry. (II) to determine the function of TGF-beta IR, IIR, Smad2, Smad3 and Smad4 signaling in regulating first branchial arch morphogenesis during craniofacial development using gain-of-function (transgenic overexpression) and loss- of-function (antisense) experiments. (III) to determine the impact of TGF-beta signaling on the expression of Lef1 and assess the morphological changes caused by altered Lef1 expression. These studies will identify and determine the critical molecules in TGF-beta signaling pathway and how TGF-beta signaling regulates the expression of Lef1 during the specification and morphogenesis of tooth, Meckel's cartilage and tongue. Ultimately, this study will contribute to our understanding of how the TGF-beta signaling cascade regulates normal craniofacial development and how disruption in TGF-beta signaling pathway can lead to craniofacial malformations.